Proliferation and Differentiation PDGF has been associated with mitogenesis and chemotaxis in osteoblasts. While PDGF has been linked to activation of phosphatidylinositol-3-kinase signal transduction pathway in fibroblasts, hepatocyte, and platelets, this growth factor has only been shown to stimulate phospholipase C and the canonical phosphatidylinositide pathway in osteoblasts. The purpose of this project is to determine whether PI-3-Kinase is involved in the proliferation and differentiation of osteoblasts when subjected to PDGF. Murine osteoblast-like cells, MC3T3-E1, will be exposed to PDGF at various concentrations and time intervals. These cells will then be lysed and immunoprecipitation will be performed with various antibodies against proteins of the PI-3-Kinase pathway. Western blots will be constructed to analyze the proteins of the immunoprecipitation and PI-3-Kinase assays will be performed to show that these proteins are associated with this signal transduction pathway. Since protein complexes form between the various subunits of the PI-3-Kinase pathway, our goal is to map this pathway in the osteoblasts from receptor to nucleus and cellular activity. Thus far, we have found the PI-3-Kinase is indeed activated in MC3T3-E1 osteoblasts when subjected to concentrations of PDGF ranging from 5-100ng/m1. One to ten minute PDGF exposures produce maximum PI-3-Kinase activity. Wortmannin, an irreversible inhibitor of the p110 subunit of PI-3-Kinase, was found to inhibit PDGF-induced PI-3-Kinase activity of MC3T3-E1 cells both in vivo and in vitro. In order to determine the physiological role of PI-3-Kinase in osteoblasts, we are initially examining chemotaxis and alkaline phosphatase as indicators of differentiation. PDGF stimulates chemotaxis in MC3T3-E1 cells. When these osteoblasts are exposed to Wortmannin, chemotaxis is significantly reduced, indicating that the PI-3-Kinase signal transduction pathway is necessary for initiation of locomotion. Experiments to determine whether PI-3-Kinase functions to regulate differentiation of osteoblasts through evaluation of alkaline phosphatasein the presence and absence of Wortmanmin are presently being conducted. EGF, insulin, and calcium have also been found to stimulate the PI-3-Kinase pathway in MC3T3-E1 osteoblasts. In the future, we hope to determine what aspects of osteoblast differentiation are controlled by these growth factors/agents via the PI-3-Kinase pathway.